On interaction with the intestine, the mycotoxin ochratoxin A (Maresca et al. 2001) is known to cause rapid inflammation, diarrhoea and increased bacterial translocation. All these effects are consistent with a decrease in epithelial barrier function, but this has not been shown directly. In this study we demonstrated that ochratoxin A is able to reduce the barrier properties of the model intestinal cell line CaCO-2.
CaCO-2 cells were seeded on Transwell cell culture inserts with a mean pore size of 0.4 µM (Costar) at 3 X 105 cells cm-2 and were grown for 21 days or until the transepithelial electrical resistance (TER) had become stable. TER was monitored using an Evometer (World Precision Instruments) fitted with Chopstick electrodes. TER was normalised by the area of the monolayer and the background TER of blank filters was subtracted from the TER of the cell monolayer. Permeability measurements were made using membrane-impermeant FITC-dextrans. Dextrans were added to the apical chamber and basal chamber fluid collected after 4 h incubation and analysed using a fluorimeter. Tight junction protein constituents were analysed by immunoblotting. Statistical analysis was performed using the non-parametric Mann-Whitney test. Values given are means ± standard deviation.
We monitored the TER of CaCO-2 cells growing in the presence or absence of 100 µM ochratoxin A over 24 h. In untreated cells the TER remains stable over this time course at around 3200-3300 V cm2 When 100 µM ochratoxin A was added to either the apical or basolateral chamber, the transepithelial resistance of CaCO-2 monolayers reduced by approximately 40 % (± 6 %) in 24 h, with significant differences between control and treated cells observed as early as 4 h post-treatment (P < 0.04, n = 6). At the same time, the permeability of the monolayer increased by approximately 2.5-fold with respect to 4 and 10 kDa FITC dextrans. In contrast no change in the permeability of either 20 or 40 kDa dextran (n = 5) was observed. When tight junction protein constituents were analysed by immunoblotting, a reduction in the levels of claudin isoforms 3 and 4, but not 1 was observed.
These results suggest that ochratoxin A is able to modulate the paracellular pathway in CaCO-2 cells by removal of specific claudin isoforms.
This work was funded by the Digestive Diseases Foundation